IL-2 and related cytokine systems are being studied to clarify the immune response in normal, neoplastic, and immunodeficient states. Following T-cell activation by antigen, the magnitude and duration of the T-cell immune response is determined by the amount of IL-2 produced, levels of receptors expressed, and time course of each event. The IL-2 receptor contains three chains, IL-2Ra, IL-2Rb, and gc. Dr. Leonard cloned IL-2Ra in 1984, and his lab co-discovered IL-2Rb in 1986 and then reported in 1993 that mutation of the gc chain results in X-linked severe combined immunodeficiency (XSCID, which has a T-B+NK- phenotype) in humans. We reported in 1995 that mutations of the gc-associated kinase, JAK3, result in an autosomal recessive form of SCID that phenocopies XSCID and in 1998 that T-B+NK+ SCID results from mutations in the IL7R gene. Based on work in our lab and others, gc was previously shown to be shared by the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. We previously cloned the IL-21 receptor, generated IL-21 transgenic mice and IL-21R knockout mice, elucidated the mechanism of IL-21 signaling, showed with Dan Littman (NYU) that IL-21 can promote the differentiation of Th17 cells (which mediate pathological processes such as Crohn's disease and psoriasis), and critically regulates immunoglobulin production. We and others showed IL-21 serves key roles in autoimmune disease, including lupus and type 1 diabetes, and indicated the possible utility of IL-21 as an anti-tumor agent. We also previously analyzed the role of IL-21 related to the development of T follicular helper cells and Th17 cells and generated data in a collaborative study that IL-21 is anti-tolerogenic cytokine in the late-phase alloimmune response. We also previously found that IL-21 signaling is required for CD8 T cell survival and memory cell formation in response to vaccinia infection and contributed to a study showing that IL-21 was pivotal in determining age-dependent immune responses in a mouse model of hepatitis, a finding that may explain why decreased production of IL-21 in younger patients may prevent critical CD8 T and B cell responses, with viral clearance in most adults and chronic HBV in neonates and children. We previously also found that IL-21 promotes the pathogenic response to pneumonia virus of mice (PVM), a viral related to human respiratory syncytial virus, and showed that after infection of Il21r-deficient mice with PVM, there was less infiltration of neutrophils and fewer CD8, CD4, and gamma-delta T cell numbers in the lungs than in WT mice. These data indicated that IL-21 plays a role in mediating the inflammatory response to PVM and suggest that inhibiting IL-21 might represent a mechanism for treatment PVM and potentially other viral infections. We previously showed that IL-21 can induce IL-10 expression and then with Tom Tedder that IL-21 could furthermore expand B regulatory cells that produce IL-10 (B10 cells). Previously, we demonstrated that IL-21 regulated expression of the Prdm1 gene that encodes BLIMP1 via a response element that depends on STAT3 and IRF4, and this led to our finding that in contrast to its ability to cooperate with PU.1 in B cells to act via Ets-IRF composite elements (EICEs), IRF4 cooperates with BATF/JUN family proteins to act via AP1-IRF composite elements (AICEs) in T cells, as well as in some B cells. We demonstrated critical regulation of important genes via these AICEs and demonstrated cooperative binding of IRF4, BATF, and JUN family proteins, with markedly diminished IRF4 binding in Batf-deficient cells and markedly diminished BATF binding in Irf4-deficient cells. We then extended studies, including contributing to studies related to IRF8 in collaboration with the lab of H.C. Morse. IL-21 has broad actions on T- and B-cells, but its actions in innate immunity are poorly understood. Previously, we reported that IL-21 induces apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). Importantly, the canonical pathway for IL-1 production requires TLR-mediated NF-kB-dependent Il1b gene induction, followed by caspase-containing inflammasome-mediated processing of pro-IL-1. We also previously showed that IL-21 unexpectedly induces IL-1 production in conventional dendritic cells (cDCs) via a STAT3-dependent but NF-kB-independent pathway. IL-21-induced IL-1 processing in cDCs does not require caspase-1 or caspase-8 but depends on IL-21-mediated death and activation of serine protease(s). Moreover, STAT3-dependent IL-1 expression in cDCs at least partially explains the IL-21-mediated pathologic response occurring during infection with pneumonia virus of mice. We also previously showed that IL-1 receptor signaling is critical for the development of autoimmune uveitis, a process we previously showed was dependent on IL-21. Mechanistically, although IL-21 can activate several STAT family transcription factors, we showed that in addition to its mainly signaling via STAT3, STAT1 is also important and that Tbx21 and Ifng were differentially regulated by these two STATs, with opposing actions of STAT1 and STAT3 on IFN- expression in CD4(+) T cells during chronic lymphocytic choriomeningitis infection, and moreover, concordant actions of IL-21 related to IFNG and TBX21 expression was found using CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, a disease caused by STAT3 mutations, as well as in cells from STAT1 gain-of-function patients. Thus, STAT1 vs. STAT3 activation is a mechanism of fine tuning the actions of IL-21. We also previously demonstrated opposing roles for IL-21 and IL-2 in T helper 9 cell differentiation, and we contributed to studies showing that IL-21 interacts with IFNg and IL-4 to govern TBET and CD11c expression in TLR-activated B cells, and that IL-21 signaling in B cells but not T cells is essential for the development of collagen-induced arthritis in mice. Moreover, we have continued a collaboration with Dr. Brian Annex related the loss of IL-21 receptor activation in hypoxic endothelial cells impairing perfusion recovery after hindlimb ischemia and that there is up-regulation of endothelial IL-21 receptor in peripheral artery disease. In the current review year, in a series of collaborations, we have advanced the studies of IL-21. In particular, we reported with Brian Kelsall's group that the defective IgA to atypical intestinal commensal bacteria in Il21r-deficient mice changes immune cell homeostasis and immunity, we assisted Xiaoxia Li's lab in their demonstration that Act1, an adaptor for IL-17 receptors, is a negative regulator in T and B cells by inhibiting STAT3 and that deficiency of Act1 results in hyper-STAT3 activation in response to IL-21. Moreover, in this setting, IL-21 improves the Sjogren's and SLE-like diseases in the Act1-deficient mice. Finally, we defined and reported a study analyzing IL-2-STAT5 versus IL-21-STAT3 based super-enhancers and elucidated aspects of cytokine-mediated gene regulation. In additional ongoing work, we are studying the roles of IL-21 in other immunological models, including related to the innate immune system, and generated a large amount of new data on signaling by IL-21 and elucidating novel actions of this cytokine. Overall, these studies have elucidated the biology and mechanisms of action by the gc family cytokine IL-21.They have expanded our knowledge of normal and pathological immune cell function and are relevant to autoimmunity and cancer, as well as to the basic control of T-cell and B-cell actions.